Recombinant Human G-protein coupled receptor 87 (GPR87)

What is GPR87 and what family of proteins does it belong to?

GPR87 is a member of the G protein-coupled receptor (GPCR) superfamily, which represents the largest protein family encoded by the human genome. GPCRs are characterized by their seven transmembrane spanning domains and function as signal transducers that convert extracellular stimuli into intracellular responses . GPR87 has been identified as a lysophosphatidic acid (LPA) receptor . The GPCR family is divided into classes A (rhodopsin), B (secretin and adhesion), C (glutamate), and F (Frizzled) subfamilies based on amino acid sequences . GPR87 belongs to the class A (rhodopsin-like) family, which is the largest and most studied group of GPCRs.

What is the three-dimensional structure of GPR87?

The 3D structure of GPR87 has been modeled using threading methods with templates such as 2VT4 and 2ZIY, which share approximately 21% sequence identity with GPR87 . The modeled structure reveals seven transmembrane helices (7TM) and 8 small α-helices . A key structural feature is the DRY-motif (Asp-Arg-Tyr sequence) located at the end of transmembrane helix 3, which is critical for GPCR binding and signal transduction . Validation of the GPR87 model using Ramachandran plots, DOPE scores, and tools like Verify-3D, ProSA, and ERRAT has confirmed the stereochemical quality of the predicted structure . Molecular dynamics simulations have been performed to analyze GPR87 binding in the presence of explicit solvent using the CHARMm force field .

How is GPR87 expression regulated?

GPR87 expression is regulated through several mechanisms:

• Transcriptional regulation: Signal transducer and activator of transcription 3 (STAT3) directly binds to STAT3-responsive elements (SREs) in the GPR87 promoter to increase its expression . Specifically, STAT3 binds to the first SRE (SRE1) in the GPR87 promoter, as demonstrated by chromatin immunoprecipitation (ChIP)-qPCR assays .

• Stimulation by cytokines: Treatment with IL-6, which activates STAT3, dramatically increases GPR87 expression at both mRNA and protein levels .

• p53-dependent regulation: GPR87 is upregulated by p53 and by DNA damage in a p53-dependent manner .

• Histone variant H3.3: Recent research indicates that H3.3 serves as an activator for GPR87 transcription, with H3.3 gene expression positively correlating with GPR87 gene expression .

What cancer types show significant GPR87 overexpression?

GPR87 has been found to be overexpressed in multiple cancer types:

• Lung cancer: Approximately 70% (7/10) of lung cancer cell lines show GPR87 overexpression . It is particularly overexpressed in lung squamous cell carcinoma and adenocarcinoma .

• Pancreatic ductal adenocarcinoma (PDA): GPR87 serves as an independent prognostic factor for PDA patients, with high GPR87 expression correlating with poor outcomes .

• Hepatocellular carcinoma (HCC): GPR87 promotes growth and metastasis in HCC, particularly in CD133+ cancer stem-like cells .

• Other cancers: GPR87 overexpression has been documented in breast, bladder, and urethral epithelial tumors .

What signaling pathways does GPR87 activate in cancer cells?

GPR87 activates several key signaling pathways in cancer cells:

• JAK2/STAT3 pathway: In pancreatic ductal adenocarcinoma, GPR87 forms a positive feedback loop with JAK2 and STAT3. GPR87 activates JAK2, which in turn activates STAT3. STAT3 then binds to the GPR87 promoter to increase its expression, completing the loop . This mechanism promotes the expansion of PDA stem cells.

• AKT-eNOS-NO axis: In lung adenocarcinoma, GPR87 promotes invasiveness and metastasis through the AKT-eNOS-NO signaling axis .

• NF-κB pathway: In pancreatic cancer, GPR87 enhances aggressiveness by activating the NF-κB signaling pathway .

• KRAS and c-Myc expression: Silencing of GPR87 results in significant decreases in KRAS and c-Myc expression, suggesting that GPR87 may regulate these oncogenic factors .

How does GPR87 promote cancer stem cell expansion?

GPR87 promotes cancer stem cell (CSC) expansion through several mechanisms:

• In pancreatic ductal adenocarcinoma (PDA), GPR87 significantly enhances sphere formation ability, increases side population (SP) cell numbers, and increases the expression of PDA stem cell markers (CD133, EpCAM, CD24, CD44, and MET) .

• GPR87 increases tumor initiation ability in vivo. In limiting dilution experiments, even 1,000 GPR87-overexpressing cells could generate tumors, while an equivalent number of GPR87-knockdown cells failed to do so .

• The positive feedback loop between GPR87, JAK2, and STAT3 plays a crucial role in promoting PDA stem cell expansion. Inhibiting JAK2 activation in GPR87-overexpressing PDA cells significantly inhibits the expansion of PDA stem cells .

• In hepatocellular carcinoma, GPR87 overexpression up-regulates CD133 expression (a marker of cancer stem cells) and enhances CSC-related properties . This correlation is particularly significant in HCC tissues with intrahepatic metastasis .

What methods are used to detect and measure GPR87 expression?

Several methods are commonly used to detect and measure GPR87 expression:

• Quantitative real-time PCR (qRT-PCR): This technique is used to measure GPR87 mRNA expression levels. In studies, qPCR has been employed to analyze the effect of GPR87 on the expression of cancer stem cell markers and to validate changes in GPR87 expression following various treatments .

• Western blot analysis: This protein detection method is used to evaluate GPR87 protein expression levels. Western blots can confirm findings at the mRNA level and examine relationships with downstream signaling molecules .

• Immunohistochemistry (IHC): IHC is used to detect GPR87 protein expression in tissue specimens and to analyze correlations between GPR87 expression and clinical parameters or other molecular markers .

• Microarray analysis: Gene expression data for GPR87 can be extracted from platforms such as the Agilent-014850 Whole Human Genome Microarray. These data are available in public repositories like the Gene Expression Omnibus (GEO) database under specific accession numbers (e.g., GSE47460) .

How can GPR87 function be modulated in experimental settings?

GPR87 function can be modulated through several experimental approaches:

• Overexpression systems:

  • Lentiviral vectors (e.g., lenti-GPR87) can be used to stably overexpress GPR87 in cell lines .

  • Transfection of GPR87 expression plasmids into appropriate host cells can allow for the study of GPR87 in isolation .

• Gene silencing techniques:

  • Short hairpin RNA (shRNA) targeting GPR87 can be delivered using adenoviral vectors (e.g., Ad-shGPR87) to downregulate GPR87 expression .

  • siRNA transfection can be used for transient knockdown of GPR87 .

• Pharmacological modulators:

  • Since GPR87 is a lysophosphatidic acid (LPA) receptor, LPA can be used as an agonist .

  • JAK2/STAT3 pathway inhibitors like AG490 can indirectly affect GPR87 function by disrupting the GPR87-JAK2-STAT3 positive feedback loop .

• Site-directed mutagenesis:

  • This technique can be used to identify binding sites for ligands and important residues for coupling to G-proteins .

  • Modifications of substrate ligands to form polar interactions with specific residues (e.g., Arg115 and Lys296) can be explored for potential inhibition .

What in vivo models are appropriate for studying GPR87 function?

Several in vivo models have been used to study GPR87 function:

• Orthotopic mouse models:

  • NOD/SCID mice with orthotopically inoculated GPR87-overexpressing or control cells (e.g., SMMC-7721-lenti-GPR87) in the liver have been used to study the effect of GPR87 on tumor growth and metastasis .

• Subcutaneous xenograft models:

  • Nude mice with subcutaneously implanted human cancer cells (e.g., H358 cells) have been used to evaluate the antitumor effects of GPR87 inhibition .

• Limiting dilution experiments:

  • Injection of varying numbers of GPR87-overexpressing or knockdown cells (100,000, 10,000, or 1,000 cells) can assess tumor initiation ability and stem cell properties .

• Pulmonary fibrosis models:

  • Mouse models of pulmonary fibrosis have been used to study the role of GPR87 in fibrotic diseases, with gain-of-function and loss-of-function experiments demonstrating pro-fibrotic and anti-fibrotic effects, respectively .

• Ex vivo models:

  • Precision-cut lung slices (PCLS) from human tissue have been used to study GPR87 in the context of fibrosis induction .

How does GPR87 interact with the JAK2/STAT3 pathway?

The interaction between GPR87 and the JAK2/STAT3 pathway involves a sophisticated positive feedback loop:

• STAT3 as an upstream regulator: Gene Set Enrichment Analysis (GSEA) reveals that GPR87 expression positively correlates with STAT3-regulated gene signatures in pancreatic ductal adenocarcinoma (PDA) . STAT3 directly binds to STAT3-responsive elements (SREs) in the GPR87 promoter, particularly the first SRE (SRE1), as demonstrated by ChIP-qPCR assays . Treatment with IL-6, which activates STAT3, significantly increases GPR87 expression at both mRNA and protein levels .

• GPR87 as an activator of JAK2/STAT3: Conversely, GPR87 activates Janus kinase 2 (JAK2), which subsequently activates STAT3 . In GPR87-overexpressing cells, the phosphorylation levels of both JAK2 and STAT3 are significantly increased, indicating enhanced activation of this pathway .

• Breaking the feedback loop: Inhibiting JAK2 activation using the JAK2/STAT3 pathway inhibitor AG490 in GPR87-overexpressing PDA cells significantly inhibits the expansion of PDA stem cells . This confirms the functional importance of this pathway in mediating GPR87's effects.

• Clinical correlation: In PDA specimens, GPR87 expression positively correlates with the phosphorylation levels of STAT3 (p = 0.002, r = 0.832) and JAK2 (p = 0.028, r = 0.718) . This correlation is observed in both tissue microarrays and freshly collected PDA specimens, validating the existence of this feedback loop in clinical samples.

What is the relationship between GPR87 and the AKT-eNOS-NO signaling axis in lung cancer?

In lung adenocarcinoma, GPR87 promotes invasiveness and metastasis through the AKT-eNOS-NO signaling axis:

• GPR87 overexpression in lung adenocarcinoma: Studies have shown that GPR87 expression is upregulated in lung adenocarcinoma and is associated with poor patient prognosis .

• Activation of AKT: GPR87 overexpression leads to the activation of AKT (protein kinase B), a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes including cell proliferation, apoptosis, and migration .

• Stimulation of eNOS: Activated AKT phosphorylates endothelial nitric oxide synthase (eNOS), increasing its activity .

• NO production: Activated eNOS catalyzes the production of nitric oxide (NO), a signaling molecule that can promote various aspects of cancer progression including angiogenesis, invasion, and metastasis .

• Metastatic phenotype: Through this AKT-eNOS-NO signaling axis, GPR87 overexpression promotes the invasiveness and metastasis of lung adenocarcinoma cells both in vitro and in vivo .

This signaling pathway represents a distinct mechanism from the JAK2/STAT3 pathway observed in pancreatic cancer, highlighting the context-dependent nature of GPR87 signaling across different cancer types.

What is the role of GPR87 in pulmonary fibrosis?

Recent research has identified a significant role for GPR87 in pulmonary fibrosis:

• Expression pattern: GPR87 is highly expressed in aberrant basaloid cells (ABC) or aberrant basal-like cells (AbBaC) in idiopathic pulmonary fibrosis (IPF) lungs but is rarely found in disease-free lungs .

• Clinical correlation: GPR87 expression in IPF is associated with more severe disease based on analyses from the Lung Genomics Research Consortium (LGRC) data .

• Genetic links: Rare variants of GPR87 (p.X359E and c.842–845del) were found to segregate in two small kindreds with familial pulmonary fibrosis, suggesting a potential causative role .

• Experimental evidence:

  • In vitro stimulation of epithelial cells

  • Ex vivo induction of fibrosis in human precision-cut lung slices (PCLS)

  • In vivo mouse models of fibrosis

All these experimental approaches consistently showed high GPR87 expression in fibrotic conditions .

• Functional effects:

  • Gain-of-function experiments demonstrated pro-fibrotic effects

  • Loss-of-function experiments in vitro, ex vivo, and in vivo blunted fibrosis

• Mechanistic link: As a lysophosphatidic acid (LPA) receptor, GPR87 may be mechanistically linked to fibrosis through LPA signaling, which has been implicated in pulmonary fibrosis pathogenesis .

This emerging role of GPR87 in pulmonary fibrosis expands its significance beyond cancer and identifies it as a potential therapeutic target for fibrotic lung diseases.

What approaches have been used to target GPR87 for cancer therapy?

Several approaches have been explored for targeting GPR87 in cancer therapy:

• Gene silencing strategies:

  • Short hairpin RNA (shRNA) delivered via adenoviral vectors (Ad-shGPR87) has effectively downregulated GPR87 expression in cancer cells .

  • This approach significantly inhibited cell proliferation in GPR87-overexpressing lung cancer cell lines (H358 and PC9) and exerted significant antitumor effects against GPR87-expressing H358 xenografts in vivo .

• Disruption of downstream signaling:

  • Inhibitors of the JAK2/STAT3 pathway, such as AG490, have been used to disrupt the GPR87-JAK2-STAT3 positive feedback loop in pancreatic cancer, significantly inhibiting the expansion of cancer stem cells .

• Structure-based drug design:

  • Computational analysis of the 3D structure of human GPR87 protein has implications for structure-based drug design .

  • In silico modification of substrate ligands to form polar interactions with specific residues (Arg115 and Lys296) has been explored as a strategy to develop potential GPR87 inhibitors .

• Targeting LPA-GPR87 interaction:

  • As GPR87 functions as a lysophosphatidic acid (LPA) receptor, approaches targeting the LPA-GPR87 interaction are being investigated, particularly in the context of pulmonary fibrosis .

How effective is GPR87 silencing in preclinical models?

GPR87 silencing has shown promising effectiveness in preclinical models:

• In vitro efficacy:

  • Ad-shGPR87 effectively downregulated GPR87 expression and significantly inhibited cell proliferation in GPR87-overexpressing lung cancer cell lines (H358 and PC9) .

  • GPR87 knockdown in pancreatic ductal adenocarcinoma cells significantly reduced sphere formation ability, decreased side population cell numbers, and decreased the expression of cancer stem cell markers .

• In vivo efficacy:

  • Treatment with Ad-shGPR87 exerted significant antitumor effects against GPR87-expressing H358 xenografts in nude mice .

  • In limiting dilution experiments, GPR87 knockdown cells showed significantly reduced tumor initiation ability compared to control cells. While 1,000 control cells could generate tumors, an equivalent number of GPR87 knockdown cells failed to do so .

  • The volume of tumors generated from GPR87 knockdown cells was significantly smaller than those from control cells .

• Molecular effects:

  • GPR87 silencing resulted in significant decreases in KRAS and c-Myc expression, suggesting disruption of key oncogenic pathways .

  • In pulmonary fibrosis models, loss of function experiments with GPR87 blunted fibrosis in vitro, ex vivo, and in vivo .

These preclinical findings collectively suggest that GPR87 silencing could be an effective therapeutic strategy for GPR87-overexpressing cancers and potentially for fibrotic diseases.

What are the challenges in developing GPR87-targeted therapies?

Despite promising preclinical results, several challenges exist in developing GPR87-targeted therapies:

• Structural complexity:

  • Like other GPCRs, GPR87 has a complex three-dimensional structure with seven transmembrane domains, making it challenging to develop small molecule inhibitors with high specificity .

  • The binding site for endogenous ligands is deep within the transmembrane helices, further complicating drug design .

• Context-dependent signaling:

  • GPR87 activates different signaling pathways in different cancer types (JAK2/STAT3 in pancreatic cancer, AKT-eNOS-NO in lung cancer) , suggesting that therapeutic approaches may need to be tailored to specific cancer types.

• Delivery mechanisms:

  • Gene silencing approaches such as shRNA require effective delivery systems that can target tumor cells specifically. The development of such delivery systems remains a significant challenge in cancer therapy .

• Resistance mechanisms:

  • Cancer cells often develop resistance to targeted therapies through various mechanisms, including activation of alternative signaling pathways. The potential resistance mechanisms to GPR87-targeted therapies have not been fully elucidated.

• Patient selection:

  • Identifying patients who would benefit most from GPR87-targeted therapies requires reliable biomarkers. While GPR87 overexpression itself could serve as a biomarker, additional predictive biomarkers may be needed to optimize patient selection.

• Potential side effects:

  • The role of GPR87 in normal physiology is not fully understood. Targeting GPR87 could potentially lead to adverse effects in tissues where it plays important physiological roles.